Accurate mass determination is a technology in which a mass of an ion is determined with a mass spectrometer with an accuracy of 1/106, that is, an accuracy at ppm level, and an elemental composition of the ion is determined based on this accurate mass. Structural elucidation of a sample molecule is carried out from the determined elemental composition of the ion. Since a molecular formula is directly determined, this technology also makes a significant contribution to accurate identification and analysis of molecular structure of an unknown component. Mass spectrometers that can perform accurate mass determination are a double-focusing magnetic sector mass spectrometer, a time-of-flight mass spectrometer (so-called TOF), and the like.
In particular, TOF that has been developed includes a quadrupole-TOF (Q-TOF) arranged with two quadrupole mass spectrometers (QMS) and an ion trap-TOF in which an ion trap composed of a ring electrode and a pair of endcap electrodes and a TOF are combined. The use of these TOFs enables accurate mass determination in an ordinary mass spectral measurement.
An example of Q-TOF is disclosed in JP-A No. 154486/1999 (Patent Document 1), and that of ion trap-TOF is disclosed in JP-A No. 123685/2003 (Patent Document 2).
In the accurate mass determination with TOF, work of mass calibration for values obtained with the instrument becomes necessary for improvement of accuracy.
When a singly-charged ion with a mass of M is accelerated with an acceleration voltage U, the ion flies through vacuum at a velocity v. The velocity v is evaluated by the following equation:v=1.39*104√(U/M)  (1)
When the ion is assumed to require a time t (second) to fly through the flying space of TOF with a length of L (meter), t is determined by Equation 2 below.t=L/v=L/(1.39*104√(U/M))=k√(M)  (2)
where k is a characteristic constant of the instrument. Namely, the flight time t of the ion is proportional to the square root of its mass. In a practical TOF instrument, the relationship between the flight time of the ion, i.e. the detection time t of the ion, and M is approximated by the following equation:M=at2+bt+c  (3)where a, b, and c are constants. That is, a quadratic equation is derived for the relation between the ion mass M and the detection time t. The process to determine the relation (Equation 3) is mass calibration.
In the mass calibration, a standard material that gives a plurality of ions with known masses is introduced into TOF, followed by measurement of mass spectrum. The constants, a, b, and c in Equation 3 can be determined using the detection times t of the product ions and the known masses M. Accordingly, a substance that gives ions with known masses in a wide mass range is used for the standard material.
After completing the mass calibration, a practical sample is measured, and the mass M0 of the sample ion can be determined from the detection time t0 of the ion according to Equation 3. The mode in which the mass calibration with the standard material and the measurement of the practical sample are independently carried out with a time interval is called external reference method. An example of the external reference method is disclosed in JP-A No. 74697/2001 (Patent Document 3).
In general, the accuracy of mass determined by this external reference method is, however, only at most 30 to 100 ppm (ppm=10−6). The poor accuracy is caused by drifts in expansion and contraction of flight space L in TOF due to changes in temperature surrounding the instrument and the like, and by drifts in an acceleration voltage U, an electrostatic lens voltage, and the like. With such accuracy, it is not possible to determine an elemental composition univocally from the determined mass M.
The determination of elemental composition with little possibility of error requires accuracy of 5 ppm or lower. In order to insure this level of accuracy in the determination, it is necessary to inject ions of a standard material together with those of an analyte sample into TOF and measure them at the same time. Ions arising from the standard material have known masses, and these ions are called lock mass ions. Generally, this method is called internal reference method. This internal reference method allows not only temperature drift and the like to be compensated but also determination to be always performed with high accuracy. Furthermore, the internal standard material injected with the analyte sample into an ion source of TOF is not required to generate ions in a wide mass range, thereby facilitating selection of the standard material. An example of the internal reference method is disclosed, for example, in JP-A No. 28252/2001 (Patent Document 4).
Hence, the internal reference method is a method essential for improvement in accuracy of determination. However, in the case of TOF that has a function of performing MS/MS such as Q-TOF in which two QMSs are arranged in pre-stages of TOF, mass calibration by means of the internal reference method cannot be applied for accurate mass determination of product ions obtained by MS/MS analysis. This is because, when a precursor ion is isolated in a first QMS, the lock mass ions of a standard sample having been introduced with an analyte sample are removed by the first QMS and are not introduced into the TOF with the product ions at the same time. Owing to the lack of the lock mass ions in the mass spectrum of the product ions, accurate mass determination by the internal reference method becomes impossible.
An example to address this problem by focusing on a precursor ion during MS/MS is disclosed in Journal of American Society for Mass Spectrometry, 10 (1999), 1305–1314 (Non-patent Document 1). Specifically, the procedure is as follows:
Accurate mass determination of an unknown sample is carried out in advance by an ordinary method (determination without MS/MS measurement), and the accurate mass of an ion to be selected for the precursor ion is determined. Then, MS/MS measurement is performed for the selected precursor ion (isolation of the ion, collision-induced dissociation (CID), measurement of product ions), and the precursor ion slightly remaining on the mass spectrum of the product ions is used as the lock mass ion, thereby carrying out mass calibration of the product ions.
[Patent Document 1] JP-A No. 154486/1999
[Patent Document 2] JP-A No. 123685/2003
[Patent Document 3] JP-A No. 74697/2001
[Patent Document 4] JP-A No. 28252/2001
[Non-patent Document 1] Journal of American Society for Mass Spectrometry, 10 (1999), 1305–1314
In the above method according to Non-patent Document 1, however, accurate mass determination of an unknown sample must be performed first by an ordinary MS mode. Then, various parameters of Q-TOF are changed to switch to a MS/MS mode, followed by MS/MS measurement. That is, the ordinary accurate mass determination and the MS/MS measurement must be conducted separately at a time interval. This method is considered as a kind of the external reference method, and thus highly accurate determination is difficult because error is duplicated. Moreover, when a plurality of unknown analyte samples are injected into the mass spectrometer one after another in a short time as in the case of LC/MS analysis, it is difficult to apply the method of Non-patent document 1.
Although Q-TOF allows MS/MS measurement, there is no report, other than Non-patent document 1, that is concerned with a mode to perform accurate determination of product ions in MS/MS measurement. Furthermore, though Q-TOF allows MS/MS measurement, it is not possible to perform MSn measurement that enables higher structural information to be acquired. Naturally, accurate mass determination by MSn is impossible with Q-TOF.